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22대 대한민국 국회 국회정보길라잡이 국회의원검색
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ABSTRACT 10

I. 서론 12

II. 재료 및 방법 18

1. 균주 18

2. 기본배양 18

1) 보존 배지 18

2) 실험 배지 18

3) 대식세포 19

3. 방법 19

1) 접종용 현탁액 19

2) 배양 19

3) 균사체 생장 및 균체외 다당체 측정 20

4) 영양 성분에 따른 큰 느타리버섯의 최적배지 조성 21

5) 셀레늄 첨가에 따른 큰 느타리버섯의 생장 및 laccase activity 23

6) 큰 느타리버섯의 균체외 다당류의 대식세포 활성 25

III. 결과 30

1. 영양성분에 따른 큰 느타리버섯의 균사체 생장, 균체외 다당체 생산 및 laccase activity 30

1) 탄소원에 따른 영향 30

2) 질소원에 따른 영향 33

3) Oil 첨가에 따른 영향 36

4) pH의 영향 39

5) 최적배지에서의 pH, 균체외 다당체의 변화 및 laccase activity 43

6) 최적 배지에서 배양 시간에 따른 큰 느타리버섯의 laccase 활성 48

2. 셀레늄 첨가에 따른 큰 느타리버섯의 생장 및 laccase activity 50

1) 큰 느타리버섯의 생장에 미치는 셀레늄의 영향 50

2) 셀레늄 첨가에 따른 배지 내 환원당 및 단백질의 변화 52

3) 셀레늄 첨가에 따른 배지 내 laccase activity 55

4) Confocal Microscopy 56

5) Scanning Electron Microscopy(SEM) 58

6) 셀레늄 첨가에 따른 laccase gene 발현 60

3. 큰 느타리버섯의 균체외 다당류의 생리 활성 63

1) Insoluble-glucan과 soluble-glucan의 물리적 특성 63

2) RAW 264.7 cell 에서의 면역활성 67

3) Insoluble-glucan과 soluble-glucan의 cytokine gene 발현에 미치는 영향 71

IV. 고찰 82

참고문헌 87

국문요약 94

List of Tables

Table 1. Composition of Raper's medium 18

Table 2. Description of primers for qRT-PCR 29

Table 3. Effect of carbon sources on the fermentation of P. eryngii 31

Table 4. Effect of nitrogen sources on the fermentation of P. eryngii 34

Table 5. Effect of various oils on the fermentation of P. eryngii 37

Table 6. Composition of IPPE and SPPE obtained from culture medium of P. eryngii. 63

Table 7. FT-IR spectra of the IPPE and SPPE obtained from the culture of P. eryngii. 66

List of Figures

Fig. 1. Fruit bodies of P. eryngii 17

Fig. 2. Mycelia of P. eryngii 17

Fig. 3. Purification procedure of mycelial and exo-polysaccharide from P. eryngii cultures. 20

Fig. 4. Effects of different carbon sources on laccase activity by the mushroom P. eryngii. Laccase activities were measured after 7 day culture. 32

Fig. 5. Effects of several nitrogen sources on laccase activity by the mushroom P. eryngii. Laccase activities were measured after 7 day culture. 35

Fig. 6. Effects of different lipid sources on laccase activity by the mushroom P. eryngii. Laccase activities were measured after 7 day culture. 38

Fig. 7. Effects of initial pH on variation of culture pH during submerged fermentation of the P. eryngii. 40

Fig. 8. Effects of initial pH on the mycelial growth of P. eryngii in cultures at 28 ℃, 160 rpm, 7 days. The medium contained glucose 2 %, yeast extract 0.4 %, K₂HPO₄0.1 %, KH₂PO₄0.046 %, MgSO₄·7H₂O 0.05 %, and corn oil 0.25 %. 41

Fig. 9. Effect of initial pH on the mycelial growth and EPS production P. eryngii. The cultivation was performed 28 ℃, 160 rpm, 7 days in medium contained glucose 2 %, yeast extract 0.4 %, K₂HPO₄0.1 %, KH₂PO₄0.046 %, MgSO₄·7H₂O... 42

Fig. 10. pH changes on commercial additives in P. eryngii culture media (culture conditions; 28℃, 160 rpm, 10 days, and initial pH 6.0). The medium contained glucose 2%, yeast extract 0.4%, K₂HPO₄0.1 %, KH₂PO₄0.046 %, MgSO₄·7H₂O 0.05%, corn... 44

Fig. 11. Effect of commercial additives on the mycelial growth and EPS of P. eryngii in cultures at 28℃, 160 rpm, 10 days, initial pH 6.0. The medium contained glucose 2%, yeast extract 0.4%, K₂HPO₄0.1 %, KH₂PO₄0.046 %, MgSO₄·7H₂O 0.05%, corn... 45

Fig. 12. Changes of pH(■) and EPS(◆) in optimal medium. Medium contained 2% glucose, 0.4% yeast extract, 0.1% K₂HPO₄,0.046% KH₂PO₄, 0.05% MgSO₄·7H₂O and 0.25% oil and initial pH 6.0. The cultivation was at 28℃, 160 rpm for 10 days on a... 46

Fig. 13. Effects of different Industrial sources on laccase activities by the mushroom P. eryngii. 47

Fig. 14. Optimal condition on laccase activity by the mushroom P. eryngii. Laccase activities were measured at pH 6.0 after 7 day culture. 49

Fig. 15. Effects of selenium concentration on the mycelial growth of P. eryngii. 51

Fig. 16. Effects of selenium concentration on the reduce sugar of P. eryngii. 53

Fig. 17. Effects of selenium concentration on the protein and ammonia of P. eryngii. 54

Fig. 18. Effects of selenium concentration on the laccase activity of P. eryngii. 55

Fig. 19. Confocal image of P. eryngii mycelia cultured in a liquid medium on selenium concentration, illustrating the different images produced by four culture medium. 57

Fig. 20. Morphology of the Mycelium of P. eryngii. 59

Fig. 21. Effect of selenium on peroxidase VPL1 gene expression in P. eryngii. 61

Fig. 22. Effect of selenium on laccase(ery3) gene expression in P. eryngii. 62

Fig. 23. FT-IR spectra of IPPE and SPPE obtained from the culture of P. eryngii. 65

Fig. 24. Effect of IPPE and SPPE obtained from P. eryngii on production of nitric oxide in a macrophage cell line. LPS treatment at concentratoin 1 ㎍/㎖. 68

Fig. 25. Effect of IPPE and SPPE isolated from P. eryngii on cytotoxicity in a macrophage cell line. LPS treatment at concentratoin 1 ㎍/㎖. 70

Fig. 26. ELISA plate for measuring TNF-α concentration. 72

Fig. 27. Effect of IPPE and SPPE from P. eryngii on TNF-α production of RAW 264.7 cell. 73

Fig. 28. Total RNA analysis by Bioanalyzer(agilent). 75

Fig. 29. IL-1β gene expression induced by IPPE and SPPE in RAW 264.7 cell. LPS treatment at concentratoin 1 ㎍/㎖. Data are presented as relative change (fold) compared to untreated control cells. 77

Fig. 30. IL-12α gene expression induced by IPPE and SPPE in RAW 264.7 cell. LPS treatment at concentratoin 1 ㎍/㎖. Data are presented as relative change (fold) compared to untreated control cells. 79

Fig. 31. TNF-α gene expression induced by IPPE and SPPE in RAW 264.7 cell. LPS treatment at concentratoin 1 ㎍/㎖. Data are presented as relative change (fold) compared to untreated control cells. 81

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